Patients with Type 2 diabetes have insulin resistance, which continues to manifest as hyperglycemia and results in the cells failure to absorb insulin. Insulin resistance in the brain affects cognitive abilities such as learning, memory and also alters synaptic plasticity. The purpose of this study is to investigate cognitive impairments in rodent models of T2 diabetes. Minimal doses of streptozotocin (STZ), which is toxic to insulin producing beta cells, were given for 9-10 weeks. Once glucose levels reached above 250mg/dl which is considered indicative of a hyperglycemic state similar to Type 2 diabetes injections would stop. Weights were takes consistently for appropriate injection volume. We utilized a spatial working memory task known as delayed alternation to test cognitive impairments. We found a significance between control and experimental rats in working memory accuracy using a one-way ANOVA p=.05. Similar to diabetic patients, we saw weight fluctuations in experimental rats, but weights were stable for the control group. This type of task requires strong working memory demands on subjects, which may be compromised by a hyperglycemic state. Additionally, previous research shows that elevated glucose levels and chronic neuroinflammation are also found in Alzheimer's disease (AD) patients. Neuroinflammation might be a pathological similarity in the progressions of Type 2 diabetes and AD.

According to the Center for Disease Control, more than one third of the U.S population is obese. Preliminary data in our laboratory has shown that obesity reduces the number of T cells in the intestines of mice. This reduction of T cells leads to impaired tissue repair in dextran sulfate sodium (DSS)-induced colitis models and compromises the protective barrier against pathogens and exposure from the outside environment. Thus, our goal was to identify how to restore the number of T cells to improve intestinal homeostasis. We investigated whether T cell numbers in the intestine can be fully restored through diet-induced weight loss. Briefly, 6 week old male mice were placed in one out of three cohorts: one cohort was placed on a normal chow diet for 14 weeks, the second cohort was placed on a high fat diet for 14 weeks, and the third cohort was placed a high fat diet for 7 weeks and switched to a normal chow diet for the remaining 7 weeks. While mice placed on a high fat diet exhibited a significant reduction in T cell number, mice that underwent diet-induced weight loss exhibited a full restoration of T cell number in the small intestine. In addition, the dieted mice also showed improved survival when challenged with DSS-induced colitis. These studies are now being followed up with RNA-sequencing experiments to investigate how gene expression is changing within the intestine and better understand the mechanisms of T cell loss in obesity.

Impella devices provide temporary mechanical circulatory support (MCS) by means of a catheter consisting of a left ventricular (LV) inlet, a microaxial pump within the catheter, and an outflow port in the proximal aorta. The recommended position of the inlet is 4-4.5cm below the aortic valve annulus. Following initial placement, the device is prone to migration, potentially leading to ineffective support, hemolysis, ventricular arrhythmia, or mitral regurgitation. Positioning is typically monitored by echocardiography. However, the Impella device has a bend between the inflow and outflow ports, and traditional 2D-imaging may not be reliable in assessing the location of the device, as single plane 2D windows may not include both the inflow port and the aortic annulus, leading to foreshortening of the cannula and under-measurement of the distance. We hypothesized that 3D echocardiography provides more accurate characterization of the Impella cannula location. We analyzed 25 echocardiograms of patients with Impella devices placed in LV which had analyzable 2D and 3D images. Measurements of the distance from the aortic annulus to the inflow port were made according to recommendations from established measurement guidelines. Three sets of measurements were made at end systole and at end diastole. Full volume 3D datasets acquired from the parasternal window were acquired an analyzed using Qlab software to overcome possible foreshortening of the Impella cannula. In addition, the 3D dataset was used to measure the angle deviation of the cannula from the long axis of the left ventricular outflow tract. There was no recognizable benefit of 3D techniques in measuring aortic annulus to Impella inflow port distance. A larger sample size may be necessary to detect a significant difference. 3D imaging may have a benefit in observing other relationships, such as the mitral apparatus, and bears ongoing investigation.

In vitro aptamer ribosensors use small molecule binding kinetics to sensitively discriminate between concentrations of a target ligand. Ribosensor devices have been used to quantify metabolite production for metabolic engineering but are also potentially useful for fields such as point-of-care diagnostics. To be useful in this field, further understanding of the parameter space in which these devices work is required. Two big unknowns within the parameter space are how different media conditions and how the use of lyophilized T7 RNA polymerase affect ribosensor function. To evaluate the potential of ribosensors in point-of-care applications, we measured RNA aptamer ribosensors with in vitro fluorescence assays of the ribosensors in both synthetic urine and its control media. Lyophilizing the T7 polymerase allows for easier transport of heat-sensitive proteins; enabling the use of ribosensors as shelf-stable molecular diagnostics even in low resource clinical care settings. We demonstrate the effects of different factors during lyophilization on the function of aptamer ribosensor devices and optimized lyophilization conditions for optimal function from the reconstituted enzymes. These optimized conditions utilize cryoprotectants to ensure that the T7 polymerase maintains its stability during and after the lyophilization process. Cryoprotectants that have been found to best stabilize T7 polymerase are the addition of sucrose solution and liquid nitrogen flash freezing. This project clarifies the conditions that are essential in transitioning the ribosensor devices from the laboratory to point-of-care diagnostics. We show how these new conditions affect the ability of the ribosensor devices to make accurate measurements. Future work will aim to modify the design for the RNA aptamer ribosensors to function more optimally within the point-of-care parameter space.

In the context of the cell, molecular crowding can be described as the summation of molecular forces acting on polypeptides at the high protein concentrations that exist naturally in membrane-bound structures. This phenomenon of molecular crowding can affect protein conformations and interactions, particularly for proteins that require high flexibility to recognize and bind a large number of interactors like the chaperone Hsp90. To observe changes in protein conformations and interactions, we cross-link proteins with lysine-reactive linker molecules, then identify and quantify cross-linked sites using mass spectrometry. The linkage sites provide physical distance constraints useful for structural predictions, and the observed changes in cross-link abundance levels is informative of conformation and interaction changes. In this project, we apply chemical cross-linking to proteins under conditions that exert different levels of molecular crowding. These conditions include in vivo cross-linking of in-tact cells where high protein density causes high levels of crowding, and cross-linking of cellular lysates where protein density and molecular crowding effects are reduced. This presentation will highlight cross-linked peptides in Hsp90 and how these are being used to improve our understanding of the effects of molecular crowding on the function of this important chaperone.

Non Hodgkin Lymphoma (NHL) affects over 70,000 people in the United States each year. Inappropriate activation of B-cell receptor signaling is associated with NHL, and therapies that target this pathway are showing promising results in the clinic. One example is ibrutinib, a small molecule inhibitor of Bruton’s Tyrosine Kinase (BTK). While ibrutinib has improved the prognosis for the majority of NHL patients, many patients eventually develop resistance to ibrutinib due to mutations in BTK and its downstream target Phospholipase C gamma 2 (PLCG2). Thus, it is important to understand how these mutations affect cell signaling, with the ultimate goal of designing new strategies to mitigate ibrutinib resistance. Previous studies in the James Lab suggest that long-term treatment of cultured cell lines with ibrutinib leads to increased cellular reactive oxygen species (ROS). However, it is unclear if ROS contribute to ibrutinib resistance or if they are a byproduct of other mechanisms that drive resistance. I hypothesize that cells with mutations commonly seen in ibrutinib-resistant patients may also exhibit increased ROS, and these cells may be sensitive to small molecules that regulate ROS. In order to test this hypothesis, I used CRISPR/cas9 gene editing with homology-directed repair to generate lymphoma cell lines that express a leucine-to-phenylalanine mutation in PLCG2. This is a well-described mutation in ibrutinib-resistant patients. Preliminary analysis shows that these mutant cells are resistant to ibrutinib. I measured ROS in these cells by ROS-Glo luminescence. Then, I cultured the cells with ROS-regulating compounds including SOD-1 and NADPH oxidase inhibitors to assess how ROS affect their viability. My goal is to clarify the role of ROS in the acquisition of ibrutinib resistance. These studies may provide justification for further pre-clinical evaluation of small molecule regulators of ROS in lymphoma, especially among subjects with acquired ibrutinib resistance.

Photovoltaic (PV) electricity generation has become much cheaper in recent years and as a result is becoming a larger percentage of total energy production. However, growth is limited due to the high capital expenditure (CAPEX) required to build new PV factories with current technologies. Solution processing techniques (spray coating, roll-to-roll, etc.) to deposit thin-film absorber materials such as CuIn(S,Se)₂ and Cu(In,Ga)(S,Se)₂ represent a much lower CAPEX alternative to current PV processes. Using simple metal chloride precursor salts dissolved in polar aprotic solvents, our group has shown solar power conversion efficiencies of 13.4%, which is a world-record for solution processed CIS. Understanding the complexation chemistry of precursor salts in solution is essential to making stable solutions which produce homogeneous absorber layers after thermal annealing. Using solubility experiments and calorimetry to examine interactions between the precursors in anhydrous dimethylformamide (DMF), we were able to infer participating molecules and stoichiometry of the complexes formed in solution between the metal chlorides, thiourea, and solvent molecules. We have also made CIS absorbers under various thermal annealing conditions, and studied the resulting changes in 1) elemental composition profiles using glow-discharge optical emission spectroscopy (GDOES) and energy-dispersive X-ray spectroscopy (EDX) and 2) film morphology using scanning electron microscopy (SEM). These results represent steps forward in improving solution processing techniques for low CAPEX solar cell manufacturing which will increase the prevalence of renewable energy to combat global warming.

The vestibular system is integral to reflex behaviors that allow humans to walk and see clearly. For example, the angular vestibulo-ocular reflex (aVOR) stabilizes images on the retina by using input from the semicircular canals to produce fully compensatory rotation of the eye. When these systems fail, patients are disabled. Understanding the vestibular system can help us create effective treatments for vestibular disorders. Our lab is developing a vestibular neural prosthesis based on such knowledge. In this proposal, we are studying large gaze shifts accomplished with eye and head movements. The eyes saccade to a target as the head starts to rotate. During this combined movement, ratio between head velocity and compensatory eye counter rotation (VOR gain) is continually adjusted to accomplish an accurate gaze shift. When the eyes move toward the target, the VOR may be reduced or eliminated. When the eyes reach the target, the gain returns to 1.0 to stabilize the eye on the target. Our study utilizes head free Rhesus monkeys to assess the gain of the VOR before, during, and after a gaze shift. The monkeys are trained to follow a laser target, allowing researchers to induce gaze shifts by changing the laser position. We utilize only horizontal shifts, and only stimulate the horizontal canal. We record change in eye velocity in response to implant stimulation at different trial times. We hypothesize that the gain of the VOR is decreased when the eyes and head are moving toward the target. By stimulating before, during, and after and evaluating the change in eye velocity, we can infer the magnitude of change induced by vestibular input and the instantaneous gain of the VOR. The results of this study will provide insight into how the brain utilizes vestibular information during combined head and eye movements.

Interactions between HIV-1 and dendritic cells (DCs) have been suggested to play a role in HIV-1 pathogenesis. DCs are antigen presenting cells that take up, process, and present foreign material to T-cells. Previous research suggests that HIV-1 can exploit this process by binding to DCs, allowing HIV-1 virions to relocate to lymph nodes where infection of T-cells can occur. HIV-1 mediates this interaction through binding of the HIV-1 envelope glycoprotein (Env) to DC-SIGN, a glycan binding protein located on the surface of DCs. Env contains a high amount of oligomannose glycans, enabling Env to act as a binding partner for DC-SIGN. Due to high sequence variation, HIV-1 strains exhibit variability in glycan presentation. A gap remains in our knowledge of where DC-SIGN binding occurs and how glycan presentation across various isolates modulates these interactions. In this study, we applied a combination of biophysical and structural approaches to characterize Env:DC-SIGN interactions across multiple HIV-1 strains. Biolayer interferometry experiments measuring binding affinity demonstrate that DC-SIGN binds with strong affinity towards Env from different isolates. This suggests that DC-SIGN is capable of recognizing different oligomannose glycan presentations and may not have a well defined epitope. However, preliminary results using electron microscopy illustrate that Env glycoprotein structure can be disrupted and altered via binding to DC-SIGN. This appears to be an isolate-specific response, as some strains remain unchanged. Using single particle approaches, we identified potential epitopes in heavily glycosylated regions of Env where additional densities are attributed to DC-SIGN. We infer that in addition to binding HIV Env, DC-SIGN may shield key neutralizing epitopes within these glycosylated regions. Our results provide further insight into the interactions that occur between HIV Env and DC-SIGN, and suggest a mechanism where HIV-1 hijacks DC’s normal immune function allowing HIV-1 trafficking while shielding neutralizing epitopes.

This research aims to determine the difference between exosomes isolated from different cell culture assays. Exosomes are cell-derived extracellular particles that are present in all eukaryotic fluids and different cell culture methods may result in exosomes with different properties. This project seeks to quantify the differences between exosomes isolated from cells cultured in 2D tissue culture plates versus cells cultured in 3D porous polymer scaffolds, and these differences include: size of exosomes, exosomes yield per cell, outer membrane markers on exosomes (CD9, CD81), and total RNA contained within the exosomes. T-75 cell culture plates and poly(hydroxyethyl methacrylate) pHEMA porous scaffolds with 40 and 100 micrometer pore size are used as 2D and 3D cell culture assays, respectively, for culturing RAW 264.7 (murine macrophage) cells. Exosomes are harvested along with cell culture medium and isolated using a total exosome isolation kit. Mean particle size and exosomes concentration are measured using a NanoSight instrument and cell concentration is measured using XTT cell viability assay. Total RNA and protein contained in exosomes are isolated using total exosome RNA and protein isolation kit, and concentration of each is measured through NanoDrop spectrophotometer. The outcome of this project not only will provide a better understanding on different properties of exosomes and the effect of cell culture assay on exosomes, but also has the potential to benefit research in tissue engineering in the future.

Each year, approximately one million medical implant-associated infections occur, due to the susceptible nature of biomaterials to microorganism colonization. In an age where antibiotic resistant strains are becoming an epidemic, it is essential to develop biomaterials that do not pose an additional risk to a patient’s health. The implantation of tissue regenerative scaffolds into the body causes a foreign body response to occur, involving the encapsulation of the device, which results in macrophage differentiation. Previous research has only focused on the tissue regeneration aspects of scaffolds, but have failed to address concerns with scaffold associated infections and immune response modulation. There is a need to further understand the role of scaffolds in modulating macrophage phenotype and the effect of macrophage differentiation towards specific phenotypes on their ability to resist microbial invasions (i.e. their phagocytic abilities). We are quantifying the differentiation of macrophages into M1 (pro-inflammatory) versus M2 (anti-inflammatory) phenotypes then comparing their phagocytic abilities using an in vitro phagocytosis assay and flow cytometry. Depending on scaffold properties (pore size, construction material), implants may promote the differentiation of macrophage towards a phenotype with diminished phagocytic abilities. By quantifying macrophage differentiation and innate immune response, we can design scaffolds that will better aid the body in healing and reduce a patient’s risk of a medical device-associated infection.

Color is often a very intentional and comprehensive choice as a visual element; artists, filmmakers, designers, and animators recognize color as a tool that signficantly shifts a viewer's perception of their work. While there are universal associations of solid colors- blue for sadness, and red for love, for example, the vastness and complexity of the color wheel leaves much room for nuance between the relationship of humans and color. Color in animation makes use of this nuance, but with a more varied stylistic choice and a greater control over very specific palettes. Through the conceptualization and production of a series of short animation clips, I aim to use color as a deliberate element to evoke subtle shifts in mood, as well as further explore the relationship between animation and color psychology. I've taken inspiration from color scripting in films through the use of a specific color palette to achieve visual balance and strong story support. My work, through tradittional and digital animation methods, aims to bring color in harmony with motion and composition to create a unique visual experience. I hope for my project to contribute to the growing understanding of color as an artistic, technical, and psychological asset in the viewer's engagement in animated films.